R E S EARCH ART I C L E

RHEUMATO ID ARTHR I T I S

Citrullinated peptide dendritic cell immunotherapy inHLA risk genotype–positive rheumatoidarthritis patientsHelen Benham,1,2* Hendrik J. Nel,1* Soi Cheng Law,1* Ahmed M. Mehdi,1* Shayna Street,1

Nishta Ramnoruth,1 Helen Pahau,1 Bernett T. Lee,3 Jennifer Ng,1 Marion E. G. Brunck,1

Claire Hyde,1 Leendert A. Trouw,4 Nadine L. Dudek,5 Anthony W. Purcell,5 Brendan J. O’Sullivan,1

John E. Connolly,3 Sanjoy K. Paul,6† Kim-Anh Lê Cao,1 Ranjeny Thomas1*‡

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In animals, immunomodulatory dendritic cells (DCs) exposed to autoantigen can suppress experimental arthritisin an antigen-specific manner. In rheumatoid arthritis (RA), disease-specific anti–citrullinated peptide autoanti-bodies (ACPA or anti-CCP) are found in the serum of about 70% of RA patients and are strongly associated withHLA-DRB1 risk alleles. This study aimed to explore the safety and biological and clinical effects of autologous DCsmodified with a nuclear factor kB (NF-kB) inhibitor exposed to four citrullinated peptide antigens, designated“Rheumavax,” in a single-center, open-labeled, first-in-human phase 1 trial. Rheumavax was administered onceintradermally at two progressive dose levels to 18 human leukocyte antigen (HLA) risk genotype–positive RApatients with citrullinated peptide–specific autoimmunity. Sixteen RA patients served as controls. Rheumavaxwas well tolerated: adverse events were grade 1 (of 4) severity. At 1 month after treatment, we observed a re-duction in effector T cells and an increased ratio of regulatory to effector T cells; a reduction in serum interleukin-15(IL-15), IL-29, CX3CL1, and CXCL11; and reduced T cell IL-6 responses to vimentin447–455–Cit450 relative to controls.Rheumavax did not induce disease flares in patients recruited with minimal disease activity, and DAS28 decreasedwithin 1month in Rheumavax-treated patients with active disease. This exploratory study demonstrates safety andbiological activity of a single intradermal injection of autologous modified DCs exposed to citrullinated peptides,and provides rationale for further studies to assess clinical efficacy and antigen-specific effects of autoantigen im-munomodulatory therapy in RA.

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INTRODUCTION

Rheumatoid arthritis (RA) is a common chronic, systemic inflamma-tory disease, leading to significant pain, disability, premature mortal-ity, and individual and societal economic burden (1). It is currentlyincurable and is managed with drugs that nonspecifically modulatethe immune inflammatory response [disease-modifying anti-rheumaticdrugs (DMARDs) and newer biologic (bDMARDs)]. Early interven-tion and combination therapy are established standards of care in RA.In themost intensivelymanaged early arthritis clinics, reported remis-sion rates range from30 to 65% (2). Adverse events limit current treat-ments, and incomplete response and loss of efficacy are common. Forexample, nonspecific suppression of inflammation or immunity is as-sociated with an increased risk of infection. Individualization of therapyto the autoimmune pathogenesis, genetic background, and relevant

1University of Queensland Diamantina Institute, Translational Research Institute, PrincessAlexandra Hospital, Woolloongabba, Queensland 4102, Australia. 2University of Queens-land School of Medicine, Brisbane, Queensland 4102, Australia. 3Singapore ImmunologyNetwork, Agency for Science, Technology and Research, 8A Biomedical Grove, ImmunosBuilding, Level 3, Biopolis, 138673 Singapore, Singapore. 4Department of Rheumatology,Leiden University Medical Center, Leiden 2333, Netherlands. 5Department of Biochemistryand Molecular Biology, School of Biomedical Sciences, Monash University, Clayton,Victoria 3800, Australia. 6Queensland Clinical Trials & Biostatistics Centre, School ofPopulation Health, The University of Queensland, Brisbane, Queensland 4006, Australia.*These authors contributed equally to this work.†Present address: Clinical Trials & Biostatistics Unit, QIMR Berghofer Medical ResearchInstitute, Brisbane, Queensland 4006, Australia.‡Corresponding author. E-mail: ranjeny.thomas@uq.edu.au

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autoantigens of RApatientsmay improve outcomes and reduce adverseevents with greater specificity.

A number of immunotherapeutic approaches have been developedon the basis of sub-immunogenic delivery of autoantigens, includingcollagen,methylated bovine serumalbumin (mBSA), and citrullinatedfibrinogen in animal models, resulting in antigen-specific deletion ofeffector T (Teff) cells and/or induction or expansion of regulatory T (Treg)cells (3–5). In RA, oral delivery of various autoantigens, including col-lagen, human cartilage gp39, and dnaJp1, has been trialed, with the aimof sub-immunogenic presentation by mucosal dendritic cells (DCs)(6–8). DCs are professional antigen-presenting cells that migrate fromperipheral tissues to draining lymph nodes, where they present antigensin a human leukocyte antigen (HLA)–restricted manner to T cells (9).Animal experiments demonstrate that delivery of immunomodulatoryDCs exposed to autoantigen can suppress experimental arthritis in anantigen-specific manner (10–12). However, translation to clinical trialsis challenging because multiple methods have been shown to modulateDC function and T cell responses, andmultiple autoantigens have beendescribed in RA, to which T cell responses are difficult tomeasure in vitro.Furthermore, the scope of what can be achieved in a clinical trial of DCimmunotherapy is limited by cost and safety considerations.

The strongest RA genetic association maps to a shared conservedregion of HLA-DRb 70–74 termed the “shared susceptibility epitope(SE),”which includesHLA-DRB1*04:01, *04:04, *01:01, and *04:05mol-ecules (13, 14). Disease-specific autoantibodies specific for citrullinatedpeptide antigens (ACPA or anti-CCP) are found in the serum of about70% of RA patients and are strongly associated with HLA-DRB1 SE

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alleles (15). Consistent with the proposed autoimmunemechanism, syn-ovial biopsies of ACPA-positive patients demonstrate high levels of lym-phocytic infiltration (16). Furthermore, circulating autoreactive T cellsrecognizing citrullinated autoantigens were identified in the pe-ripheral blood (PB) ofHLA-DRB1 SE+ RA patients with in vitro assaysor peptide–HLA-DR tetramers (17–19). Structural studies demon-strated that citrulline is accommodated for presentation within the P4pocket of HLA-DRB1*04:01 and 04:04 (20, 21). These data suggest thatACPA+HLA-DRB1SE+RApatients are an appropriate target popula-tion for initial investigation of immunomodulatory therapy consistingof modified autologous DCs exposed to putative citrullinated auto-antigenic epitopes.

Activation of the transcription factor nuclear factor kB (NF-kB)drives the coordinate up-regulation of molecules associated with DCantigen presentation (22). Multiple genes driving NF-kB pathway ac-tivation are associated with RA susceptibility (23). Furthermore, NF-kBsubunits are overexpressed in RA synovial tissue (24, 25). DCs defi-cient in the NF-kB subunit, RelB, suppressed preexisting immune re-sponses in vivo in an antigen-specific manner through induction of asuppressive CD4+ Treg cell population (26, 27). Suppression of NF-kB,including RelB, in DCs can be achieved pharmacologically in vitrowith Bay11-7082, a specific, irreversible inhibitor of NF-kB, in miceand humans (26, 28). A single subcutaneous dose of DCs modifiedwith Bay11-7082 suppressed established arthritis in an antigen-specificmanner in mice (11).

To test this strategy in humans, we carried out an exploratoryphase 1 trial of DCs modified with Bay11-7082 and exposed to ci-trullinated peptides in ACPA+ RA patients with HLA-SE risk alleles(Fig. 1). Because efficacy and tolerability were unknown and im-mune mechanism was untested in patients, our aims were to assesssafety and to describe the immunological and clinical effects of asingle dose of autologous modified DCs and citrullinated peptides inDMARD-treated RA patients with any level of disease activity andany disease duration in a first-in-human study.

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RESULTS

PatientsWe recruited 34 ACPA+ RA patients carrying HLA-DRB1 SE alleles, 9of whomwere treated with low-dose and 9 with high-dose Rheumavax;16 controls were untreated as outlined in the flowchart (fig. S1). Baselinecharacteristics and outcomes for each group are shown in Table 1 andfor individual participants in table S1.Among those treated, 55%carriedat least one HLA-DRB1*04:01 allele and 45% carried other SE alleles.The median [interquartile range (IQR)] disease duration was 3 years(1.5 to 4 years), and the baseline disease activity score (DAS28CRP)was 2.43 (1.54 to 3.81) for the low-dose group.Median disease durationwas 2 years (1 to 4 years), and baselineDAS28was 2.2 (1.56 to 3.26) forthe high-dose group. Low baseline DAS28 are as expected for a cohortof RA patients with short disease duration treated with multipleDMARDs (29). Baseline characteristics did not differ significantly be-tween treated groups and controls (Table 1).

SafetyPatients received a single intradermal administration of Rheumavaxto the upper thigh (doses indicated in Table 1 and table S1). Predictedadverse events included injection site and draining lymph node reac-

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tions, allergy or anaphylaxis, hypoglycemia (preclinical toxicity studies),and disease flare. Observed adverse events included transient new leu-kopenia or lymphopenia in six patients, transient anemia in two, andtransient new elevation of liver transaminases in two. One patienthad a self-limited headache and one had a single fasting glucose of2.9 mMmeasured by a glucometer on the day after Rheumavax. All ad-verse events were grade 1 out of a maximum 4, and there was a com-parable frequency in low- and high-dose groups (Tables 1 and 2 andtables S1 and S2).

Effects on Teff and Treg cells, antigen-specific responses, andsystemic inflammationTo assess T cell effects, we followed changes in PB CD4+ Teff and Treg cellsby flowcytometry afterRheumavax (table S3 and fig. S2). Initial assessmentwas 6 days after Rheumavax, when effects of DCs on peripheral T cellimmunity were anticipated. We focused on changes at day 6 and1 month, which were most likely attributable to Rheumavax. Figure 2compares changes in%CD4+CD25+CD127+ Teff, CD4

+CD25hiCD127−

Treg, and the ratio of Treg/Teff in patients receiving low-dose, high-dose, or no Rheumavax over 1 month. We observed a reduction in %Teff by at least 25% in 11 of 15 patients assessed within the first monthafter Rheumavax. In controls, the % Teff decreased by≥25% in 0 of 5(P < 0.01 relative to treated patients, Fisher’s exact test; Fig. 2A).Whereasthe % Treg increased by≥25% in only 5 of 15 treated patients and in 0of 5 controls (Fig. 2B), the Treg/Teff ratio increased by≥25% in 11 of15 treated patients and in 1 of 5 controls assessed over the same timeframe, consistent with an altered balance of regulatory to effector CD4+

T cells (Fig. 2C). Changes in Teff, Treg/Teff, and disease activity in eachpatient over 6 months after Rheumavax are detailed in fig. S4. In-terindividual variability was evident. Because the timing of the peakchange in Teff or Treg/Teff ratio varied, we calculated the peak% increase(Emax) or decrease (Emin) of each change above baseline after Rheuma-vax.We observed at least one of these T cell effects in all but two treatedpatients (patients 1 and 16, fig. S4 and Fig. 2D). In controls, the medianTeff Emin was 105 (88 to 127) and Treg/Teff Emax was 96 (89 to 102).

AlthoughPB autoantigen-specific proliferative responseswereweak,we previously demonstrated interleukin-6 (IL-6) production by CD4+

T cells in response to citrullinated autoantigenic peptides in RA pa-tients (18). We assessed ex vivo antigen-specific T cell proliferative andIL-6 responses to citrullinated peptides delivered with Rheumavax andto aggrecan84–103–Cit93, which was not delivered with Rheumavax, intreated patients and controls. Stimulation indices for peptide-specificproliferative responses were generally <2, as described previously (18).We therefore compared IL-6 responses by group over 1 month beforeand after Rheumavax by linear mixed-effect (LME) modeling. Individ-ual responses varied considerably and the group × time interaction wassignificant only for IL-6 responses to vimentin447–455–Cit450, with re-duction in the Rheumavax group relative to controls (P < 0.05, withcorrection for baseline) (Fig. 3A).We compared proliferative responsesto tetanus toxoid antigen in treated patients and controls as an indicatorof immune responsiveness toward nominal foreign antigen. As dem-onstrated previously, RA patients demonstrate relative T cell anergytoward tetanus toxoid (18). There was no significant difference in theresponse of the Rheumavax-treated and control groups over the firstmonth after Rheumavax; however, the tetanus toxoid response increasedin many subjects after Rheumavax (Fig. 3B).

Initial disease activity assessment was 1 month after Rheumavax.We fitted linear regression models to identify immunologic features

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associatedwithchange inDAS28at1 month(Materials andMethods and Table 3). Af-ter FDR correction, day 6 reduction inTeff, day 6 and 1 month reduction in anti-CCP immunoglobulin A (IgA)/IgG level,and peak increase in Treg/Teff were asso-ciated with reduced DAS28 at 1 month(Fig. 4, A to C, fig. S4, and Table 3). P val-ues for Emin Teff and Emax Treg/Teff were0.01 and 0.07, respectively (R2 = 0.41 and0.24, respectively), after removing a po-tential outlier data point with a change inDAS28 of −2.79. Changes in anti-CCPIgA/IgG levels were not correlated withchanges in DAS28 in controls (P = 0.64).In contrast to the changes observed in anti-CCP level, when assessing citrullinated

peptide–specific ACPA IgG responses to peptides not included inRheumavax (table S4), we found no difference in the proportion oftreated and control patients with positive assays (fig. S5).

These features suggested systemic and immunological effects ofRheumavax.We observed that CRPwasmore likely to fall in patientsreceiving higher doses of DCs per kilogram (P = 0.016, Fig. 4D). To

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further explore anti-inflammatory effects, we fitted an LME model foreach of 106 serumprotein analytes (table S5) collected at baseline, day 6,and 1 month. Reductions in 11 proteins had significant group-timeeffects discriminating Rheumavax-treated patients from controls at1 month, correcting for baseline (table S6). The concentrations ofIL-15, CX3CL1 (fractalkine), CXCL11 (ITAC), IL-29, and peptide

Ex vivo culture with IL-4 + GM-CSF + Bay11-7082 for 48-h

3-h exposure to cit-peptides

Peripheral blood collection

RheumavaxI.D. upper thigh

injection

Month 6Follow up

D -2

Month 1Follow-up

D6 Follow-up

D2Phone

follow-up

D0Injection

Month 2Follow-up

Month 3Follow-up

Ex vivo

In vivo

A

C

B

HLA-DRB1-SE

Rheumavax

Inguinal LN

Joint

Teff

Treg

HLA type

cit-peptide

Lys+

Binding pocket

Monocyte purification autologous DC:

Rheumavax

Modified

Fig. 1. Concept and schemaof immunomodulatory therapy with DCs and citrullinated

peptides in HLA risk genotype–positivepatients with RA. (A and B) HLA-DRB1 SE+

RA patients (A) are the target population foinitial investigation of immunomodulatorytherapy of modified autologous DCs (Bexposed to putative citrullinated autoantigenic epitopes (A). HLA-DR risk allotypes bindcitrullinated self-peptides due to a criticalysine residue at position 71 of the peptidebinding groove (A). DCs generated ex vivofrom PB monocytes in the presence of theNF-kB inhibitor Bay11-7082 are exposed tocitrullinated peptides and then injected intradermally (I.D.) (B). Immune response is expected in draining lymph nodes (LN) on Tefand Treg cells, with expected impact on joinsymptoms through regulation of inflammation (A). (C) Schema for ex vivo productioninjection, and follow-up. Each follow-up assessment included clinical and laboratoryevaluation of toxicity (table S2); phonefollow-up evaluated clinical evidence of toxicity only. Assessments included clinical andlaboratory evaluation of RA disease activityusing DAS scores. At each assessment, PBmononuclear cells (PBMCs) and serum werecollected for analysis of cell populations byflow cytometry (table S3), in vitro responsesto antigens (table S4), serum analytes (tableS5), anti-CCP titer, and peptide-specificACPA (fig. S5 and table S4). Fasting insulinand glucose were measured at baseline and1 month, and patients recorded fasting glucose measurements for the first 2 days afteRheumavax.

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YYwere significantly lower inRheumavax-treated patients at 1monthbut not at baseline relative to controls (Fig. 4E).

Clinical effectsTo determine immediate clinical effect or flares, we focused on the firstmonth after Rheumavax, by dose group (Fig. 5). Among the 18 treated

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patients at baseline, 9 had a swollen joint count (SJC)≥1 (DAS28 >2.4)and thus could be evaluated for clinical improvement (“active disease”).In the low-dose group, five of nine were active. DAS28 and SJC de-creased in four of four patients evaluated at 1month. Patient 1 was eval-uated at 2 months: both DAS28 and SJC were reduced. None of theremaining four patients flared: maximum DAS28 was 2.64 at 1 month.

Table 2. Clinically relevant adverse events (treatment and possibly treatment-related). ALP, alkaline phosphatase; ALT, alanine amino-transferase; AST, aspartate aminotransferase.

Systemic classof adverse event

Adverse event

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Number ofpatients affected

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Dose group (1 = low dose,2 = high dose) and time

of adverse eventsafter Rheumavax

Blood/bone marrow

Lymphopenia (0.5 × 109 to0.9 × 109/liter)

3

1 (day 30), 2 (day 30),2 (day 90)

Leukopenia (3.9 × 109 to 3.0 × 109/liter)

3 1 (day 30), 2 (day 60), 2 (day 60)

Neutropenia (1.9 × 109 to 1.5 × 109/liter)

2 2 (day 30), 2 (day 60)

Hemoglobin (100–134 g/liter)

3 2 (days 30, 60, 90), 2 (day 90),2 (days 30, 60, 90)

Metabolic/laboratory

Glucose (2–2.9 mM) 1 2 (day1)

Hepatic

ALP (120–300 U/liter) 1 1 (day 180)

AST (36–86 U/liter)

2 1 (day 90), 1 (day 90)

Hepatic

Bilirubin (19–35 mM) 1 1 (day 180)

ALT (46–112 U/liter)

2 1 (day 180)

Musculoskeletal

Headache 1 2 (day 90)

Table 1. Demographic details of treated and control patients. DAS,disease activity score; RF,rheumatoid factor; MTX,methotrexate; SSZ,sulfasalazine;HCQ,hydroxychloroquine; LEF,leflunomide; TNFi,tumor necrosis factor inhibitor; N/A,not applicable.

Rheumavax Lowdose (n = 9)

Rheumavax Highdose (n = 9)

ControlGroup 1 (n = 11)

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ControlGroup 2 (n = 5)

Age, mean (SD)

56.8 (9) 55.1 (10.1) 57.6 (9.8) 49.8 (10.8)

Females, n (%)

5 (55) 8 (89) 6 (54) 4 (88)

Disease duration (years),median (IQR)

3 (1.5–4)

2 (1–4) 1 (1–2) 2 (1–11)

Baseline DAS,median (IQR)

2.43 (1.54–3.81)

2.2 (1.56–3.3) 2.81 (2.1–3.63) 3.29 (2.75–3.83)

RF+, n (%)

7 (78) 9 (100) 10 (91) 4 (88)

Anti-CCP+, n (%)

9 (100) 9 (100) 11 (100) 5 (100)

HLA-DR SE+, n (%)

9 (100) 9 (100) 11 (100) 5 (100)

Other treatment, n (%)

MTX

7 (78) 9 (100) 7 (64) 5 (100)

SSZ

2 (22) 6 (67) 5 (45) 5 (100)

HCQ

5 (56) 6 (67) 8 (73) 4 (88)

LEF

0 (0) 0 (0) 2 (18) 1 (20)

TNFi

2 (22) 0 (0) 0 (0) 0 (0)

Rheumavax

Dose/kg (range)

7.2 × 103 to 1.7 × 104 2.7 × 104 to 6.2 × 104 N/A N/A

Adverse events, n (%)

4 (44) 5 (56) N/A N/A

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In the high-dose group, four of nine were active. DAS28 decreased inall four, and SJC decreased in three and did not change in one. None ofthe remaining five patients flared: maximumDAS28 was 2.49. DAS28and SJC followedno trend among controls (Fig. 5, A andB).When eval-uated longitudinally, the active disease group had a median changein DAS28 of −0.84 (−1.52 to 0.60) at 1 month and −0.45 (−1.58 to 0.02)at 6months, whereas the inactive disease group had amedian changeof 0.48 (−0.05 to 0.61) at 1 month and 0.58 (0.03 to 1.07) at 6 monthsafter Rheumavax. The control group had a median change of 0.12(−1.64 to 1) at 1 month and −0.58 (−2.94 to 0.65) at 6 months (Fig. 5C).All groups including the control group were actively managed for RAduring this 6-month period. Neither the ratio of modified DC/DCHLA-DR nor CD40 mean fluorescence intensity (MFI) (fig. S6) wasassociated with 1-month change in DAS28 (P = 0.55 and 0.91, respec-tively). DMARDswere sometimes reduced or stopped during the follow-up period for clinical reasons (fig. S4). Clinical improvements that hadoccurred after Rheumavax were destabilized in several cases withchanges in DMARDs (for example, patients 8 and 15; fig. S4). In patient7, a knee injury precipitated a general flare (fig. S4).

DISCUSSION

Individualization of therapy to the autoimmune pathogenesis, geneticbackground, and relevant autoantigens of RA patients may improvepatient outcomes and reduce adverse events with greater specificity.Such individualized approaches apply the principles of immune toler-ance (30). However, translation to clinical trials is challenging. Effec-tive long-termdisease suppression by restoration of immune tolerance

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will likely require intervention in a multidose regimen very early afteror even before disease onset (31). Furthermore, the safety of auto-antigenic peptide immunomodulatory therapy in RA and the best ve-hicles to achieve this are as yet unknown (32). As a first step, in thisfirst-in-human phase 1 study, we wished to explore safety and immu-nological and clinical effects of a single dose of immunodulatory DCsand citrullinated antigen in a small trial of DMARD-treated HLA SE+

RA patients within the first years after diagnosis.We found that a singleadministration of 0.6 × 106 to 4.5 × 106 Rheumavax inDMARD-treatedHLA SE+ RA patients was well tolerated. We tested two dose levels: thehigh dose was chosen on the basis of themaximumpredicted yield aftervenesection of 250 ml of blood and our experience in melanoma pa-tients (33). Human studies suggest that intradermal delivery of ≤5 ×106mature DCs optimizes migration to draining lymph nodes (34). Al-though we did not carry out labeling studies of Rheumavax, we previ-ously demonstrated migration of labeled immature DCs to draininglymphnodes after intradermal delivery (35). Preparation of Rheumavaxfrom venesected blood limited DC dose delivery: two of nine patientsreceived <1 × 106 DCs, and nine of nine received <5 × 106 DCs. Fur-thermore, two patients had self-limiting anemia during follow-up, po-tentially due to previous venesection.

The relationship of change in CRP to dose suggests a systemicanti-inflammatory effect of Rheumavax. Consistent with this notion,we observed significant reductions in activated Teff cells and proin-flammatory cytokines and chemokines inRheumavax-treated patientsrelative to untreated controls. Given that DCs modified with Bay11-7082 did not secrete higher levels of anti-inflammatory cytokinesbefore administration (fig. S8) and that DCs were administered inlow numbers to a noninflamed intradermal site, we hypothesize that

Fig. 2. Changes in Teff andTreg cells in treated andcontrol individuals. (A to C) PB % CD4+CD25+CD127+

Teff (A), % CD25hiCD127−CD4+ Treg cells (B), and Treg/Teffratio (C) for individuals at baseline (month 0), 6 days,and 1 month after low- or high-dose Rheumavax, andcontrols at baseline and 1 month. (D) The maximumdecrease in%CD4+CD25+CD127+Teff andthemaximumincrease in%CD25hiCD127−/CD25+CD127+ CD4+ T cells(Treg/Teff) were calculated in each patient after Rheuma-vax relative to baseline (that is, Emax = peak value/baseline value × 100%; Emin = trough value/baseline val-ue × 100%). Emin Teff and Emax Treg/Teff values are plottedwith median and IQR. n = 9 each for low-dose (blacksymbols) and high-dose (green symbols) Rheumavax;n = 5 for controls.

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this anti-inflammatory effect relates to the rapid change in activatedTeff after treatment. In mice, we showed that Teff proliferation in re-sponse to antigen is reduced in lymph nodes draining the site ofantigen-exposed modified DCs (26). The association of reduced DAS28at 1 month, with day 6 and peak reduction in Teff supports the con-clusion that reduction in joint inflammation is related to the Teff

response. Teff may have decreased after Rheumavax by several mecha-nisms, including deletion or anergy in response to antigen recognition,regulation, or apoptosis due to reduction in homeostatic cytokines (36).In this regard, we investigated changes in a large number of serum ana-lytes without FDR correction to identify possible immune responseproteins for validation in future trials. Although it is impossible to drawfirm conclusions in this small trial, the analytes differing betweenRheumavax-treated subjects and controls are of interest. IL-15 is akey inflammatory cytokine in RA (37), which can sensitize autoreactiveT cells to citrullinated peptides (38). Thus, IL-15 reduction could arrest

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autoreactive Teff, potentially with morewidespread effects than just peptide-specific Teff. The Treg/Teff ratio increasedin treated patients associated with thereduction in Teff. The relative increasein Foxp3+ Treg cells could have resultedfrom decreased inflammatory cytokineeffects onFoxp3 expression and enhancedTreg stability in the face of fewer Teff

(39, 40). Improvements in T cell func-tion, including recall responses to anti-gen and Treg cell function, have beendescribed with TNF inhibition (41, 42).Although serumTNFwas notmeasured,we observed a reduction in several proin-flammatory cytokines and chemokinesrelative to controls. Reduction inCX3CL1has been shown to correlatewith responseto TNF inhibitors in RA patients (43).

Here, we delivered immunomodu-latory DCs and citrullinated antigen ina selected HLA at-risk population ofRA patients. Immune assays suggestedmultiple immunoregulatory and anti-inflammatory effects of Rheumavax onT cells, serum cytokines, and anti-CCPand that some of these were associatedwith clinical effects. However, the ex-ploratory studies carried out are limitedby small sample size and interindividualvariability. The results of antigen-specificassays were not conclusive. AlthoughIL-6 responses to vimentin447–455–Cit450decreased after Rheumavax relative toresponses in control subjects, citrullinatedpeptide–specific responses varied be-tween individuals. Assays of citrullinatedself-peptide responses in vitro are lim-ited by low sensitivity. Moreover, sen-sitivity to detect a consistent change inantigen-specific T cell responses amongtreated patientswould have been limited

in a single-dose trial (44). Therefore, to adequately assess antigen-specific responses, more sensitive assays, such as pHLAII tetramersor intracellular cytokine responses after in vitro restimulation, wouldneed to be carried out in trials of larger numbers of subjects givenmultiple doses of citrullinated peptide immunomodulatory therapy(45). Nevertheless, in the current trial, tetanus toxoid–specific T cellproliferative responses were not suppressed after Rheumavax, sug-gesting that Rheumavax was not broadly immunosuppressive to aforeign antigen response.

Our study has limitations. The primary outcomes of the trial weresafety and effects on immune function. For this, and logistic reasons,the trial was open-labeled and assessed only a single Rheumavax ad-ministration. The sample size and trial design were limited by cost ofproduction of autologous DCs and potential for disease flare; there-fore, we did not deliver irrelevant control antigens or unmodifiedDCs. Because open-labeled trials are subject to placebo effects, larger,

Fig. 3. Changes in citrullinated antigen-specificIL-6 responses and tetanus toxoid–specific pro-liferative responses in treated and control indi-viduals. PBMCs (2 × 105 perwell) were incubated for5 days with aggrecan84–103–Cit93, vimentin447–455–Cit450, fibrinogen a chain717–725–Cit720, and colla-gen type II1237–1249–Cit1240 (0 or 30 mg/ml) or withtetanus toxoid [4 Limes flocculation units (Lf)/ml].IL-6 production and T cell proliferation were mea-sured by cytokine bead array. The stimulation indi-ces for peptide-stimulated/unstimulated IL-6 andT cell proliferation were calculated and are plottedfor each individual at baseline (0) and 1 month.Black symbols, low dose; green symbols, high dose.

(A) Citrullinated peptide–specific IL-6. (B) Tetanus toxoid–specific proliferation. n = 17 for Rheumavax; n =10 for controls. Differences in Rheumavax-treated and control group responses over time were comparedby LME for group ×time interaction. *P = 0.02.

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placebo-controlled, double-blind studies are needed to assess clinicalefficacy of antigen-specific immunotherapeutic approaches. It is im-portant that future trials of immunomodulation develop and includemechanistic studies as well as clinical outcomes to advance the fieldfor further mechanistic understanding to underpin the developmentof antigen-specific approaches in RA. Inclusion in future trials of a

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control arm receiving immunomodulatory DCs without peptide orwith irrelevant peptide would aid interpretation of antigen specificity.Furthermore, two control groups were recruited to compare serial clin-ical and immune markers in patients who were not treated with Rheu-mavax. These comparisons may be confounded by the small numbersof patients per group with variability in disease activity and DMARDtreatments, as well as fewer post-baseline visits in the controls. For thesereasons and because immunological and clinical effects become moredifficult to attribute to a single intervention with increasing time, welimited almost all analyses to the first month after Rheumavax treat-ment and to a 1-month follow-up in controls.

Self-peptide response assays could be optimized with advances intechnology for analysis of antigen-specific T cells. The peptidesdelivered in Rheumavax would be predicted to bind the differentHLA-DR SE molecules with variable affinity based on core sequencebinding motifs (20), and indeed, fibrinogen b chain433–441 may nothave bound all the HLA-DR molecules (fig. S7). Using anchorresidue frequencies, citrullinated peptides could be further optimizedfor binding to RA-associated HLA-DR molecules.

In this preliminary study, a single intradermal injection of autol-ogous modified DCs exposed to citrullinated peptides was safe, andimmunoregulatory and anti-inflammatory effects were observed inHLA risk genotype–positive RA patients. Further studies are neededto assess clinical efficacy and associated immune effects of antigen-specific immunotherapy in RA.

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Fig. 4. Changes in T cells, proinflammatory cytokines, and anti-CCPlevels in patients treatedwith Rheumavax and controls. (A and B) The

(D) Change in C-reactive protein (CRP) at 1 month plotted relative to DCdose/kg. The regression line is shown (r2 = 0.37). Black symbols, low dose;

relationship between the change in DAS4v at 1 month after Rheumavaxwith the changes in Teff at day 6 (A) and Emax Treg/Teff (B) are plotted,showing regression lines. (C) Serum levels of anti-CCP antibodies weredetermined using the CCP3.1 enzyme-linked immunosorbent assay(ELISA) assay. The relationship between change in DAS4v at 1month afterRheumavax with the change in anti-CCP level at day 6 after Rheumavaxis plotted, showing regression line. P and R2 values are shown in Table 3.

green symbols, high dose. (E) Serum levels of IL-15, CXCL11, CX3CL1, IL-29,and PYY were determined by luminex assay at baseline, day 6 (d6), and1 month after Rheumavax (closed circles) and at a 1-month interval incontrols (open circles). Log-transformed values are plotted as median andIQR. n = 9 for controls; n = 18 for Rheumavax. Differences at 1monthwereestimatedwith theMann-Whitney test (*P = 0.046, IL-15; P = 0.028, CXCL11;P = 0.024, CX3CR1; P = 0.045, IL-29; P = 0.027, PYY).

Table 3. Linear regression analysis of change in DAS at day 30 on lab-oratory features. Features with P ≤ 0.05 (t test) are shown in the table. Theintercepts are not shown. % Foxp3+ Treg is the % of the CD4+CD25hiCD127lo

Treg cells expressing Foxp3. Emax = peak value/baseline value × 100%; Emin =trough value/baseline value × 100%. FDR, false discovery rate (calculatedusing Benjamini-Hochberg correction).

Features

Estimate P FDR R2

% Teff, day 6

0.78 0.004 0.056 0.41

% Foxp3+ Treg, day 6

−0.65 0.009 0.14 0.36

% CD16hiCD14+ monocytes, day 30

−0.66 0.02 0.42 0.35

Emin Teff

0.73 0.005 0.064 0.37

Emax Treg/Teff

−0.71 0.001 0.009 0.57

Anti-CCP IgA/IgG titer, day 6

7.42 0.02 0.046 0.42

Anti-CCP IgA/IgG titer, day 30

1.73 0.005 0.046 0.39

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MATERIALS AND METHODS

Study patientsInclusion criteria were a diagnosis of RA with symptoms for at least3 months and treated with at least one DMARD or bDMARD, positiveanti-CCP, and carriage of HLA-DRB1 SE. Exclusion criteria were seri-ous infection or major surgery within 28 days; history or family historyof atopy or positive radioallergosorbent (RAST) test; malignancy; sig-nificant cardiovascular, renal, liver, neurological, or skin disease; posi-tive hepatitis or HIV serology; pregnancy; or drug abuse. Eleven controlRA patients were recruited and studied according to the same scheduleas the 18 treated patients, except that flow cytometric analyses andT cellresponses to antigen could not be evaluated for technical reasons in thecontrols. To compare changes in cell populations longitudinally in RApatients not receiving Rheumavax, an additional five anti-CCP+, HLA-DRB1 SE+ control RA patients were recruited: flow cytometry and in vitroassays were carried out twice, at visits 1 month apart (control group 2,Table 1 and Fig. 2).

Study designBetween 10November 2009 and 31March 2011, we conducted a single-center, open-labeled, first-in-human, controlled, prospective phase 1

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trial at the Princess Alexandra Hospital,Brisbane, in which autologous modifiedDCspulsedwith four citrullinated peptideantigens, designated “Rheumavax,” wereadministered once intradermally in twogroups: a low dose of 1 × 106 DCs and ahigh dose of 5 × 106DCs (Fig. 1B, Table 1,table S1, and full protocol). RA patientswere recruited and studied according tothe protocol and statistical analysis plan,outlined in the flowchart (fig. S1).Writteninformed consent was obtained from allpatients, and the study was approved bythe human ethics committees of theMetroSouth District and University of Queens-land, Brisbane, Australia, and by the Ther-apeuticGoodsAdministration of Australiaunder theClinical TrialNotification (CTN)scheme. Patients’ safety was monitoredand assessed against specifically designedtoxicity criteria (table S2) at continuoustime points. Safety data 1 month after ad-ministration of the lowdosewere evaluatedby the Ethics Committee before commen-cing the high-dose group (table S1).

Preparation of RheumavaxIn preliminary experiments, we deter-mined that addition of Bay11-7082 tomonocyte-derived DC cultures of RApatients was immunomodulatory: Itdose dependently reduced their capacityto stimulate allogeneic T cells in mixedlymphocyte cultures (fig. S8, A and B),similar to healthy control Bay11-7082DCs (28). In these cultures, levels of HLA-

DR, CD40, CD80, and CD86 varied between DCs generated from dif-ferent RA donors. Cell surface expression of HLA-DR and CD80consistently decreased as the concentration of Bay11-7082 increased,whereas CD40 and CD86 expression varied (fig. S8C). CCR7 was ex-pressed by 100% of Bay11-7082 DCs with an intensity equivalent toDCs (fig. S8D). Cell death increased as Bay11-7082 concentrationsincreased above 3 mM, consistent with the requirement of NF-kB forcell survival. Cytokine secretion, including IL-10 by DCs and Bay11-7082 DCs, was similar, except for significantly lower secretion of TNFby Bay11-7082 DCs relative to DCs (fig. S8D).

At the preentry visit, the concentration of Bay11-7082 was titratedin monocyte-derived DC cultures to where HLA-DR expression wasreduced while maintaining DC viability relative to control DCs. In allpatients, the concentration of Bay11-7082 ranged from 2 to 2.5 mM.Before injection, modified DCs were exposed to the following pep-tides to permit presentation by HLA-DR molecules: collagen typeII1237–1249–Cit1240, fibrinogen a chain717–725–Cit720, fibrinogen bchain433–441–Cit436, and vimentin447–455–Cit450 (table S4). Allcultures met release criteria for viability and sterility. HLA-DR andCD40 MFI was used as an internal control as evidence that the Bay11-7082 had modified the DCs relative to untreated DCs. Preset releasecriteria of 40% reduction in MFI of HLA-DR and CD40 relative to

Fig. 5. Clinical effect of Rheumavax. (A) Disease activityscore (DAS4v) at baseline and 1 month after treatment inRA patients treated with low and high doses of DCs and incontrols. Patients with active disease (SJC ≥ 1) and inactivedisease (SJC=0) aredenotedby thedashed and solid lines,respectively. (B) SJC at baselineand1month after treatmentin RA patients in low- and high-dose DC groups and in con-trols. (C) Change in DAS relative to baseline for 6monthsplotted in active and inactive groups and controls. Groupeddata are displayed asmedian and IQR. n= 9 for Rheumavaxgroups;n=11 for control group.Groupswere compared ateach time point using the Kruskal-Wallis test with Dunn’smultiple comparison test: **P = 0.005, Rheumavax activeversus inactive; P = 0.033, Rheumavax active versus control;*P = 0.013, active versus inactive; P = 0.040, inactive versuscontrol. Control data not available at 2 months.

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untreated DCs were found to be too stringent in most patients [median(IQR) actual reduction inHLA-DR, 17% (10 to 33), andCD40, +5% (11to +24)]. Because the trial was exploratory and the absolute value of thisinternal control was not expected to affect safety, the trial investigatorsagreed to record the values and release the DCs for administration. Theyield ofmodifiedDCs determined the final dose delivered to the patient,andvariedbetween0.5×106 to1×106DCs (7.2 × 103 to 1.7 × 104DCs/kg)in the low-dose group and 2 × 106 to 4.5 × 106 DCs (2.7 × 104 to 6.2 ×104 DCs/kg) in the high-dose group (Table 1 and table S1). DCs wereresuspended in 0.5 ml of sterile phosphate-buffered saline for intra-dermal injection.

PeptidesCitrullinated aggrecan, vimentin, collagen type II, and fibrinogen aand b chain peptides predicted to bind the HLA-DRB1 SE weresynthesized by Auspep Pty. Ltd. to 95% purity and tested for endo-toxin and sterility, and their identities were confirmed byN-terminalsequencing. Peptides were reconstituted in water and stored at −20°C.Final dilutions were made with medium.

HLA-DR–binding assayThe method was adapted from that previously described (46). Flat-bottom 96-well plates were coated with 100 ml per well of HLA-DR–specificmonoclonal antibody L243 at 20 mg/ml in 50mM sodiumcarbonate buffer for 2 hours at 37°C. Wells were washed and blockedwith 0.2% BSA overnight at 4°C. Lysates of the homozygous Epstein-Barr virus–transformed B lymphoblastoid cell lines expressing ap-propriate DR haplotypes were prepared, and cell debris was removedby centrifugation. Lysate (100 ml per well) was added overnight at 4°C.Wells were washed, and test peptides were added at 3- to 100-foldmolar excess with biotinylated CLIP peptide and then incubatedfor 48 hours at 37°C. Plates were washed and incubated with neutra-vidin horseradish peroxidase in binding buffer for 1 hour, developedwith o-phenylenediamine dihydrochloride, and then detected in aVarioskan plate reader.

Flow cytometric analysis of PBMCsThawed PBMCs (2 × 105) were incubated for 1 hour at 37°C and thenstained with three panels of surface and intracellular markers toassess leukocyte populations (table S3, fig. S3). Antibodies weresourced from BD Pharmingen, eBioscience, BioLegend, and Invitrogen.Tetramers for CD1dwere obtained fromDaleGodfrey (MonashUniver-sity, Melbourne, Victoria, Australia). Data were collected on the Galliosflow cytometer (Beckman Coulter) and analyzed using Kaluza software(Beckman Coulter). Live cells were identified as Aqua-negative, andgates were set on the basis of isotype and FMO controls.

Luminex analysis of serumSerum samples were collected and frozen within 16 hours at −80°C.A high-throughput multiplex assay analyzed a select panel of 106analytes (table S5) detectable after thaw in human serum sampleswith Beadlyte technology (MPXHCTYO-60K; Millipore) using theBio-Plex Luminex-100 Station (Bio-Rad), as described (47). Humanbeadmates determined the levels of several cytokines with the HumanMulti-Cytokine Flex Kit using detection protocol B (Upstate) andduplicate standards. Markers were analyzed in triplicate from a 600-mlsample. Bio-Plex Manager 4.1 software with a five-parameter logisticcurve-fitting algorithm was applied for standard curve calculations.

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Anti-CCP and ACPA fine specificity assaysInitial screening of the patients was carried out by Queensland HealthPathology Laboratories using the anti-CCP2 ELISA (Axis-Shield). Anti-body reactivity against the citrullinated (Cit) and the uncitrullinatedform of linear peptides (vimentin1–16–Cit3, fibrinogen a chain27–43–Cit36,vimentin59–74–Cit64, fibrinogen b chain36–52–Cit44, and enolase5–20–Cit9) was determined by ELISA (48) (table S4). Cutoff values for theCit-specific responses were calculated as previously described (49). Seracollected at each time point from all patients were tested using anti-CCP3.1 IgG/IgA ELISA (Quanta Lite), with titration of serum andquantification based on the standard curve to amaximumof 1000 semi-quantitative units/ml.

T cell proliferative and cytokine responses in vitroPBMCs were thawed from cryopreserved samples, and then 2 × 105

PBMCs per well were incubated with tetanus toxoid (4 Lf/ml) (ChironVaccines) or aggrecan84–103–Cit93, vimentin447–455–Cit450, fibrinogen bchain433–441–Cit436, fibrinogen a chain717–725–Cit720, and collagen typeII1237–1249–Cit1240 (0 or 30 mg/ml) in completemedium (RPMI, 1× PSG,1× sodium pyruvate, 10% human serum) for 5 days. T cell proliferationwas assessed by [3H]thymidine incorporation (18). IL-2, IL-4, IFN-g(interferon-g), IL-10, IL-6, IL-17, andTNFweremeasured inday5 super-natants using BD Cytometric Bead Array Human Th1/Th2/Th17 kits(BDPharmingen). Sampleswere read on the BDFACSArray bioanalyzersystem.

StatisticsThe patients were followed longitudinally according to the flowchart(fig. S1). Not all treated subjects attended for all visits; the patterns ofmissingness were random for all study parameters. Analysis of safety,efficacy, and immunological parameters was descriptive, including in-terparticipant comparisons using nonparametric methods. Inter- andintraparticipant comparisons over time used longitudinal methods,including linear regression and linearmixedmodels. Variables collectedlongitudinally were expressed as change (D) with respect to the non-treated baseline. We determined the maximum increase or decreasein % T cell populations (Emax, Emin), as previously described (that is,Emax = peak value/baseline value × 100%; Emin = trough value/baselinevalue × 100%) (50). Differences between groups were compared usingtwo-tailed unpaired Student’s t tests, Mann-Whitney test for nonnor-mally distributed data, and Kruskal-Wallis tests with Dunn’s correctionfor multiple testing, where three or more groups were compared with asignificance level a of 0.05. Variance for nonnormally distributed datais displayed as median ± IQR, otherwise as mean ± SD.

The immune response data set includes 39 features, and the pro-teomics data set includes 116 features. The statistical analyses usedthe statistical software R, including the stats and MASS packages.Proteomics data were log-transformed, and missing values wereimputed using the NIPALS algorithm (51).

Univariate linear regression model. The immune response datawere analyzed with linear regression models. Each predictor (feature)was expressed in terms of change of expression after Rheumavax treat-ment (day 6 or 30)with respect to the nontreated baseline. The responseof interest in themodel is theDAS between day 30 (after treatment) andthe baseline, denoted by ∆DASD30 = DASD30 − DASbaseline for eachindividual.

To identify features in which changes of expression were associatedwith∆DASD30, we performed a linear regressionmodel on each feature

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separately. Those with a regression coefficient significantly differentfrom 0 are indicated in Table 3 (P < 0.05).The normality of data wasassessed using quantile-quantile plots before the application of linearregression models.

LME model. To investigate the treatment effect in serum ana-lytes and in vitro IL-6 responses to peptides at 1 month relative tobaseline, we performed an LMEmodel with time (t) and Rheumavaxtreatment (trt) as fixed effects and random intercepts. We reportfeatures with an interaction effect (t*trt) and F test P < 0.05.

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SUPPLEMENTARY MATERIALS

www.sciencetranslationalmedicine.org/cgi/content/full/7/290/290ra87/DC1Fig. S1. Flowchart of the study.Fig. S2. Gating strategy for CD4+ Teff and Treg cells.Fig. S3. Gating strategy for B cell/T cell/Tfh and NKT/DC/monocyte/NK panels shown in table S3.Fig. S4. Effects of Rheumavax on disease activity, Teff and Treg cells, tetanus toxoid immunity,and anti-CCP titer in each treated patient.Fig. S5. Proportion of each group with citrullinated peptide–specific ACPA for the duration ofthe study.Fig. S6. Ratio of HLA-DR and CD40 expression by modified DC relative to DC before administration.Fig. S7. Frequency of anchor residues from naturally eluted peptides from HLA-DRB1*01:01,DRB1*04:01, and DRB1*04:04.Fig. S8. Generation of monocyte-derived DCs from RA PB in the presence of increasing con-centrations of Bay11-7082 reduces HLA-DR expression and the capacity to stimulate allogeneicT cells.Table S1. Characteristics, clinical outcomes, and treatment side effects for the 18 studysubjects.Table S2. Rheumavax safety toxicity criteria.Table S3. Flow cytometry panels used in the analysis of PBMCs.Table S4. Peptides used in this study.Table S5. Serum analytes measured in this study.Table S6. LME model of serum analytes.Source data (excel file)

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Acknowledgments: We thank A. Casgrain who produced Fig. 1; A. Tran who carried out clin-ical assessments; D. White and P. Vecchio who assisted with trial design and management;D. Godfrey who provided CD1d tetramer; and K. Lau, L. van Toorn, N. Bobby, and E. Dugganwho provided technical assistance. Funding: Supported by National Health and Medical Re-search Council (NHMRC) grants 301244, 351439, and 569938; an AusIndustry BiotechnologyInnovation Fund grant; a Queensland Government Innovation Start-Up Scheme grant; and thePrincess Alexandra Hospital Foundation. R.T. was supported by Arthritis Queensland and anAustralian Research Council (ARC) Future Fellowship, S.S. by an NHMRC Peter Doherty Fellow-ship, and B.J.O.S. by Queensland Government Smart State Fellowship and Arthritis Queens-land. A.W.P. is an NHMRC Senior Research Fellow. L.A.T. is a recipient of a De NederlandseOrganisatie voor Wetenschappelijk Onderzoek (NWO) ZonMw Vidi grant. S.K.P. received infra-structure funding support from Therapeutic Innovation Australia. K.-A.L.C. was supported inpart by the Australian Cancer Research Foundation for the Diamantina Individualised Oncol-ogy Care Centre at The University of Queensland Diamantina Institute. Author contributions:R.T. had full access to all of the data in the study and takes full responsibility for the integrity ofthe data and the accuracy of the data analysis. Study concept and design: R.T., B.J.O.S., J.E.C., L.A.T.,S.K.P., and K.-A.L.C. Acquisition, analysis, or interpretation of data: all authors. Drafting of the man-uscript: H.B. and R.T. Critical revision of the manuscript for important intellectual content: allauthors. Obtained funding: R.T. Technical support: H.J.N., S.C.L., S.S., N.R., H.P., B.T.L., J.N., M.E.G.B.,C.H., L.A.T., A.W.P., and N.L.D. Statistical analysis and support: A.M.M., S.K.P., and K.-A.L.C. Competinginterests: R.T. has filed provisional patents surrounding technology for targeting DCs for antigen-specific tolerance and is a director of the spin-off company, Dendright, which is commercializingvaccines that target DCs to suppress autoimmune diseases in collaboration with Janssen BiotechInc. A.M.M. was supported from this commercial source. L.A.T. is a recipient of a fellowship fromJanssen Biologics. S.K.P. has acted as a consultant and speaker for Novartis and Amylin Pharmaceu-ticals Inc. and has received grants for clinical studies fromMerck, Bristol-Myers Squibb, Novo Nordisk,and Pfizer. The study funders had no role in the design or conduct of the study; collection,management, analysis, and interpretation of the data; preparation of the manuscript; or decisionto submit the manuscript for publication. The other authors declare no competing interests. Dataand materials availability: Trial registration: anzctr.org.au; identifier: ACTRN12610000373077.

Submitted 15 February 2015Accepted 24 April 2015Published 3 June 201510.1126/scitranslmed.aaa9301

Citation: H. Benham, H. J. Nel, S. C. Law, A. M. Mehdi, S. Street, N. Ramnoruth, H. Pahau,B. T. Lee, J. Ng, M. E. G. Brunck, C. Hyde, L. A. Trouw, N. L. Dudek, A. W. Purcell,B. J. O’Sullivan, J. E. Connolly, S. K. Paul, K.-A. Lê Cao, R. Thomas, Citrullinated peptidedendritic cell immunotherapy in HLA risk genotype–positive rheumatoid arthritis patients.Sci. Transl. Med. 7, 290ra87 (2015).

nceTranslationalMedicine.org 3 June 2015 Vol 7 Issue 290 290ra87 11

rheumatoid arthritis patientspositive−Citrullinated peptide dendritic cell immunotherapy in HLA risk genotype

Purcell, Brendan J. O'Sullivan, John E. Connolly, Sanjoy K. Paul, Kim-Anh Lê Cao and Ranjeny ThomasBernett T. Lee, Jennifer Ng, Marion E. G. Brunck, Claire Hyde, Leendert A. Trouw, Nadine L. Dudek, Anthony W. Helen Benham, Hendrik J. Nel, Soi Cheng Law, Ahmed M. Mehdi, Shayna Street, Nishta Ramnoruth, Helen Pahau,

DOI: 10.1126/scitranslmed.aaa9301, 290ra87290ra87.7Sci Transl Med

for RA.was safe and did not induce disease flares. These data support larger studies of antigen-specific immunotherapyand decreased production of proinflammatory cytokines compared with untreated RA patient controls. The therapy

positive RA patients had reduced numbers of effector T cells−peptide antigens. They find that HLA risk genotypeB inhibitor that have been exposed to four citrullinatedκinjection of autologous dendritic cells modified with an NF-

report a first-in-human phase 1 trial of a singleet al. risk alleles. Benham HLA-DRB1(RA), especially those with citrullinated peptides (ACPA) are found in most patients with rheumatoid arthritis−Autoantibodies to anti

Immunotherapy out of joint

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